The Mechanism Research Of AML1/ETO Suppress The Development Of T(8;21)Acute Myeloid Leukemia Through Activation Of EGR1

Objective:Acute myeloid leukemia (AML) is a large family of highly heterogeneous invasive blood tumor, mainly due to the abnormal differentiation, malignant clone proliferation and apoptosis reduced of mature myeloid cells, t (8;21)(q22, q22) is the most common chromosome translocation of AML. Translocation of AML1/ET0(+) AML is sensitive to cytarabine. The most of t (8;21) AML patients prognosis is relatively favorable. But, why the AML1/ETO (+) AML prognosis is relatively favorable is unclear so far. Here, we found that the transcription factor EGR1is regulated by the AML1/ETO fusion protein in AML1/ETO (+) AML. AML1/ETO triggers epigenetic activation of EGR1, inhibiting AML1/ETO (+) AML cells proliferation and inducing its apoptosis in t (8;21) acute myeloid leukemia. This discover closely related to the prognosis of AML1/ETO (+) AML patients.Methods:The AML1/ETO positive AML patients were screened by chip technology in this study. The specificity high expression of EGR1gene was found. According to the expression of EGR1, they were divided into two groups. The clinical prognosis were analyzed by22case of t (8;21) AML patients. EGR1mRNA and protein expression were test in HL-60, SKNO-1, SKNO-1-siAE, U937and U937-AE cell lines by qualitative and quantitative PCR and Western Blot. Through bioinformatics analysis, we found that EGR1upstream gene promoter region contains two AML1binding sites. Genomic DNA as a template, a series of truncated EGR1gene promoter sequences were amplified by PCR. We build the promoter of EGR1which mutation/missing/AML1/ETO binding sites and the luciferase expression plasmid. The expression vector containing AML1/ETO and EGR1promoter of fluorescein enzyme carrier report gene were transfect to293T cells.Fluorescein enzyme activity were detect from transfect293T cells. AML1/ETO fusion protein interact with EGR1promoter region were analyzed by Chromatin immune coprecipitation (CHIP). The methylation frequency of EGR1promoter CpG islands were detected by bisulphite genomic sequencing in cell lines and normal bone marrow. The expression of EGR1was test in SKNO-1and U937-AE cell lines after treat with acetylation enzyme inhibitors of P300(C646), histone acetylation enzyme inhibitors (histone deacetylase inhibitor, HDACi) TSA (Trichostatin A) and demethylation drugs5-Aza (5-Aza-2’-deoxycytidine). EGR1siRNA and pcDNA3.0-EGR1were transfect into AML1/ETO positive and negative cell lines respectively by transfection technology. Then, CCK8and flow cytometry were used to detect EGR1effects on cell proliferation and apoptosis. The expression of P53and PTEN protein were detect by Western Blot after pcDNA3.0-EGRl transfect into HL-60cell lines.Results:The expression of EGR1has a significantly increased in the SKNO-1, SKNO-1-Mock and U937-AE compared with HL-60, SKNO-1-siAE and U937by qualitative, quantitative RT-PCR and protein immunoblot experiments. In the293T cell transfection, AML1/ETO can raise EGRl promoter activity. Luciferase activity of mutation of AML1/ETO binding sites (EGR1-P2-M) was significantly decreased than the luciferase activity of AML1/ETO second binding sites (EGR1-P2) by dual luciferase activity tests confirmed. Chromatin immune coprecipitation experiments have established that AML1/ETO fusion protein can raise P300to the promoter region of EGR1cause histone acetylation. The methylation level of CpG islands of EGR1promoter has no obvious differences by bisulphite genomic sequencing confirmed. Kasumi (4.8%), SKNO-1(1.4%), SKNO-1-siAE (2.4%), U937(1.9%), U937-AE (0.9%) and NBM (1.4%) were test. The expression of EGR1were significantly decreased after C646(25uM,50uM) treatment in SKNO-1and U937-AE cell lines. The expression of EGR1was significantly increased after TSA (10nM,20nM) treatment in SKNO-1and U937-AE cell lines. But, the expression of EGR1was no obvious change after5-Aza (2.5uM,5uM) after reatment in SKNO-1and U937-AE cell lines. The expression of EGR1was significantly increased after TSA (10nM,20nM) and5-Aza (2.5uM,5uM) treatment in SKNO-1and U937-AE cell lines. But, it has no obvious change compared with TSA alone. The proliferation of SKNO-1which was transfect into EGR1siRNA was significantly enhanced after0h,24h,48h,72h by cell proliferation experiment. The proliferation of SKNO-1-siAE which was transfect into pcDNA3.0-EGR1was significantly inhibited after0h,24h,48h,72h by cell proliferation experiment. The apoptosis of SKNO-1-siAE which was transfect into pcDNA3.0-EGR1was significantly increased by flow cytometry. The expression of P53and PTEN protein were significantly increased after transfect with pcDNA3.0-EGR1in HL-60cell lines. The t(8;21) AML patients which have high expression of EGR1is much better than the low expression of EGR1t(8;21) patients in the survival analysis.Conclusion:AML1/ETO can combine to the promoter region of EGR1. It can raise the epigenetic modification enzyme P300to the promoter of EGR1causing histone acetylation of EGR1. Then, histone acetylation may prevent further DNA methylation. The two kinds of enzymes work synergisticall. The expression of EGR1level of AML1/ETO (+) cell lines were significantly higher compared with AML1/ETO (-) cell lines. EGR1can inhibit cell proliferation and promote cell apoptosis in t (8;21) AML cell lines. EGR1genes is a tumor suppressor gene in human t (8;21) AML. The expression of EGR1was high in t (8;21) AML patients because of histone acetylation. Then, it promote leukemia cells apoptosis and delay the progress of t (8;21) AML. Finally, it improve the prognosis of t (8;21) AML patients.